Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid (original) (raw)

The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography-mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and 8 C 27 -or C 24 -bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3␤-hydroxy-5-cholenoic acid and 3␤,7␣dihydroxy-5␤-cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7␣,12␣-dihydroxy-3-oxo-4-cholenoic acid and its 5␤-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7␣hydroxylation of cholesterol, followed by oxidation to 7␣-hydroxy-4-cholesten-3-one and 12␣-hydroxylation. Notably, 27-hydroxylation of the product 7␣,12␣-dihydroxy-4cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27hydroxylation, yielding 3␣,7␣,12␣-trihydroxy-5␤-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7␣,12␣dihydroxy-4-cholesten-3-one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative bio-Abbreviations: CA, cholic acid (3␣,7␣,12␣-trihydroxy-5␤-cholanoate); CDCA, chenodeoxycholic acid (3␣,7␣-dihydroxy-5␤-cholanoate); CsA, cyclosporin A; ODS, octadecylsilane; TEAP-LH-20, triethylaminohydroxypropyl-Sephadex LH-20; GLC, gas-liquid chromatography; GC/MS, gas chromatography-mass spectrometry; THCA, 3␣,7␣,12␣-trihydroxy-5␤-cholestanoate.